The present invention relates to a polypeptide with appetite regulating function/activity, a nucleic acid construct encoding the polypeptide and a method of producing the polypeptide. The invention further relates to recombinant vectors comprising the nucleic acid construct encoding the polypeptide, recombinant host cells comprising the nucleic acid construct or the recombinant vector, a transgenic animal or plant containing and expressing the nucleic acid construct, an appetite regulating composition comprising the polypeptide, and the use of the polypeptide for regulating appetite.
The polypeptide has appetite regulating activity/function in mammals, including humans.
It has been known that certain tumors when implanted into rats after a period of growth suddenly induce severe anorexia and adipsia (lack of eating and drinking) in the animal, whereas closely related tumor lines do not, Madsen et al., Scand. J. Clin. Invest. Supplement 220: 27-36.
The aim of this invention has been to find the factor(s) responsible for this characteristic phenotype.
Cocaine and Amphetamine Regulated Transcript (CART) was detected as one of several compounds that was selectively expressed in anorectic versus non-anorectic secondary cultures of glucagonomas. In situ hybridisation analysis of CART mRNA expression has shown a decreased level of CART mRNA in the nucleus arcuatus and nucleus paraventricularis of the rat hypothalamus following fasting. Similarly, CART mRNA in the arcuate nucleus of Zucker rats (fa/fa) was strongly decreased when compared to heterozygote controls (fa/+) as measured by in situ hybridisation. Thus, CART mRNA in the arcuate nucleus demonstrates a pattern of change inverse to that known for NPY. The latter finding provides a strong linkage between the expression of CART and biological factors involved in food intake.
A polypeptide of at least 30 amino acids was found by Spiess et al., 1981, Biochemistry 20: 1982-1988 as an HPLC peak when purifying somatostatine from sheep hypothalamus. The isolated polypeptide was the C-terminal (IPI-CART) portion of CART. However, no biological function was associated with this molecule.
The mature CART peptide has so far not been isolated and characterised. A transcript to be upregulated in rat brain after treatment with cocaine and amphetamine relating to CART was cloned. This cloning indicates that the peptide may exist in a long form consisting of 102 amino acid residues or in a short form consisting of 89 amino acid residues (Douglass, J. et al. J. Neurosci. 15, 2471-2481, 1995).
The same group found the human gene and cDNA for CART. Only the short form exists in humans (Douglass and Daoud (1996), Gene 169: 241-245).
In 1995 Amgen disclosed methods of reducing or preventing neuron degeneration and promoting regeneration and restoration of function induced by CART (WO 96/34619).
The aim of this invention has been to find the factor(s) responsible for the above described characteristic phenotype.
It has now been found that a polypeptide with the sequence SEQ ID No. 1 and fragments thereof have appetite regulating activity/function:
Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp- Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Pro-Arg-Arg-Gln-Leu-Arg-Ala-Pro-GlY-Ala - Val -Leu-Gln-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg- Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu- Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg- Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
Furthermore, it has been found that the following polypeptides have appetite regulating activity/function:
SEQ ID No. 2:
Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp- Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys- Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met- Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu- Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 3:
Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp- Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys- Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met- Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu- Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 4:
Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu- Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg- Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 5:
Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu- Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg- Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 6:
Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly- Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro- Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 7:
Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys- Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser- Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 8:
Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly- Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe- Leu-Leu-Lys-Cys-Leu
SEQ ID No. 9:
Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu- Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 10:
Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile- Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr- Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val- Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys- Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No.11:
Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile- Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr- Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val- Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys- Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
The peptides SEQ ID Nos. 5 to 11 are considered to be novel per se and are constituting a part of the invention.
In a preferred embodiment of the present invention the cysteine residues of the above peptides SEQ ID Nos. 1 to 11 are linked by disulphide bonds in the configuration I-III, II-V and IV-VI when the cysteines are numbered from the N-terminal. These peptides are also considered to be novel per se and are constituting a part of the invention.
In the present context, the term xe2x80x9cappetite regulating activity/functionxe2x80x9d is intended to mean any activity/function which suppresses appetite e.g. by inducing a feeling of satiety or by inhibiting the sensation of hunger. The appetite regulating activity/function may be measured according to the test methods described in Example 9 or 20
In another aspect, the invention relates to nucleic acid constructs comprising a nucleotide sequence encoding a CART polypeptide or a fragment or variant thereof with appetite regulating activity/function.
In a further aspect, the invention relates to nucleic acid constructs encoding a polypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to 9 such as the sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are linked by disulphide bonds in the configuration I-III, II-V and IV-VI when the cysteines are numbered from the N-terminal end.
In a further aspect, the invention relates to recombinant vectors comprising the nucleic acid constructs and recombinant host cells comprising the nucleic acid constructs or the vectors.
In a further aspect, the invention relates to a method of producing a CART polypeptide or a fragment or a variant thereof with appetite regulating activity/function which method comprises cultivating a host cell as defined above in a suitable culture medium under conditions permitting expression of the nucleic acid construct and recovering the resulting polypeptide from the culture medium/cell.
In a further aspect, the invention relates to transgenic animals or transgenic plants comprising the nucleic acid construct as defined above as well as methods of producing a CART polypeptide or a fragment or a variant thereof with appetite regulating activity/function using such transgenic animals or transgenic plants.
In still a further aspect, the invention relates to an antibody capable of specifically binding to a CART polypeptide or a fragment or a variant thereof with appetite regulating activity/function, such as a polypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to 9, e.g. the sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are linked by disulphide bonds in the configuration I-III, II-V and IV-VI when the cysteines are numbered from the N-terminal end. In a preferred embodiment of the invention the antibody is monoclonal and the invention furthermore relates to hybridomas producing such monoclonal antibodies.
In a further aspect, the invention relates to appetite regulating compositions comprising the polypeptides as defined above and a pharmaceutically acceptable carrier and the use of the polypeptides for the preparation of medicaments for the regulation of appetite. In a preferred embodiment of the invention the medicaments are used for the treatment of obesity.
Furthermore, the invention relates to a method for the regulation of appetite comprising administering to a subject in need thereof an effective amount of a polypeptide as defined above. In a further aspect, the invention relates to the use of CART or CART fragments or variants to identify a functional receptor and the subsequent use of the CART receptor to identify receptor agonists with appetite regulating activity.
In a further aspect, the invention relates to compounds that upregulate the CART expression and thereby regulate appetite.